Effects of seasonal changes on in vitro developmental competence of porcine oocytes.

Abstract

The aim of this study was to investigate whether seasonal changes affected in vitro developmental competence of porcine oocytes. The relationship between atmospheric temperature and embryonic development of in vitro matured porcine oocytes following intracytoplasmic sperm injection was examined throughout the year. The blastocyst rate (31.1%) in winter (mean atmospheric temperature during December to February: -3.8 C) was significantly higher (P<0.05) than those of other seasons in 2008/2009 (19.7-23.5%; 6.3-17.5 C). The monthly mean blastocyst rates were negatively correlated with the temperatures (r=-0.5944, P<0.05). The results of the present study suggest that porcine embryos could be produced throughout the year, but the in vitro production efficiency was significantly affected by season, i.e., atmospheric temperatures. Furthermore, the results showed that winter is a favorable season for blastocyst production in the region of Obihiro, Hokkaido, Japan.

Full text

Page 1

帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge
' '
Title
Effects of Seasonal Changes on In Vi tro
Devel opm ental Com petence of Porci ne O ocytes
Author(s)
SUZUKI, Yo, W ATAN ABE, H i royuki , FUKUI, Yutaka,
福井, 豊
Citation
J ournal of Reproducti on and Devel opm ent, 56(4):
396-399
Issue Date2010
URLhttp: //i r. obi hi ro. ac. j p/dspace/handl e/10322/2814
Rights日本繁殖生物学会

Page 2

J oum al o f Reproduction and D evel opm ent， Vol . 56， N o. 4， 2010
- Or i g i na l Ar t ic1守 一
Effects of Seasonal Changes on In Vitro Devel opm ental 仁om petence of
Porcine O ocytes
Y o SUZUKI1)，
I)D epartm ent of Food Production Science， O bihiro U nive目ity of A gricu ltu re and Veterin ary M edicine， O bihiro 080-8555
and 2)D epartm ent of Ani m al Production Science， The U nited G raduate Schoo/ of A gricu ltu ral Science， Iw ate U n iversity，
M orioka 020-8550， japan
Abstr. ct. T he . i m of 出is st udy was to investigate whet her seasonal changes affected in四trodevel opm ental com pet ence
of porci ne oocytes. The rel ati onshi p bet ween a位nospheri c tem perature and em bryoni c devel opm ent of in vitro m at ured
porci ne oocytes fol l ow i ng i ntracytopl asm i c sper m injection was exam i ned t hroughout the year. The bl astocyst rate
(31 . 1 %) in .w inter ( m ean atrnosphe口c t em perat ure duri ng Decem ber to February: -3. 8 C) was significantly hi gher
(P<0.05) t h. n those of other seasons i n 2008/2009 (19.7-23. 5% ; 6. 3-17.5 C ). The m ont hl y mean bl astocyst rates w ere
negati vel y correl ated wi t h 血e tem peratures (r=-O .5944，
P<0.05). The results of the present study suggest that po目 me
em bryos coul d be produced t hroughout the year， but the in vitro producti on efficiency was significant 1y affected by
season， i.e . ， atm ospheri c t em pera加res. Furt herm ore，正he results showed血at w i nter is a favorabl e season for bl astocyst
producti on in the regi on of O bi hi ro，
J apan
Ke y words: Em bryoni c devel opm enι Pi g，
Seasonal changes，
Hi r oyuki WA T A N A B E1， 2 ) a n d Yut aka FUKUI1)
Hokkai do，
Tem per a加 re
1
m aturation (IVM)， 晶 此 凶 凶 1 口 liz
been u ti日 liz 田ed 自
po
porcl 田 町anes a 悶 available from slaughterhouses a t any tim e of
th e year. In our laboratory，
p 町 cin e blastocysts have been produced
in v itro using defined cu ltu問 m edi a after intracytoplasm ic spenn
in jectio n (I CSI) [1-3]. A sign ifican t probl em fo r porcine I VF is a
hi gh pol ysperm y rate ( 50- 60% ) th at 問 su lts in em bryo polyp-
lo id izatio n [4，
5 ]. At p 田 sent ， it seem s 由a t ICSI is 由eonly m ethod
th at c田 be used w ith po四 in e I VP to com pletely elim inate th e risk
ofpol yspenny and produce nonnal d ip loid em b 吋 o s
Sum m er heat s t 悶 ss induces l ow developm ental c剖 npetence in
bovine em bryos and l ow conception ra回
in fertility m ay be strongly 問 lated to high a回losph町 IC 匝m pera-
tures. Som e rep町 ts show the detrim ental effects on oocyte
cytoskeletal organi 四 tio n [9 ] and m eiotic [1町 anddevelopm ental
com petence [11] caused by subjecting porcine ovaries or oocytes to
heat stre ss. On th e other hand， porcine oocyte 芯and em bryos have
high s町lsitivity to l ow脂m peratu問 s com pared w i 出 those of other
m am rr叫 ianspecies [12 ] 日nce porcine oocyt， 田 町 V町田田itive
加 extrem e tem pe問 旬 開s， 由e I VP efficiency m ay be influenced by
se田onal changes. To our know l edge， ho 附 ever， 出ee能 cts of sea-
so 川a l changes on th e efficacy of porci ne I VP have not been
in vestigated . Furtherrno田， it is not kn O ¥ '四 w hether th ere is se田 ooal
m島 氏ility in pigs in H okkai do，
a region w ith a com paratively cooler
clim a也 阻dsh orter sum m er period山田 th em ai nl and of Japan
In th e p問 sent study， we investigat吋 the effects of seasonal
川 叩p 阿 rod 削 u 叫 叫 c ω 凶 山 伽
t “由 伽 州
io
加 o
凹E
沼四
z 回a 飢 副 t“ io
凹n (IVF 町 ) and c 山u lt 加 u陪 (IVC) has
， 問 esearch on em b 耐 ぽyo 叫logy and c ∞
江 in e IVP can be pe凶耐四edthroughout th e year，
白 or r
。mme 町rc 白1 回 a 叫 1pu
町r -
田se 田s . Por 問c
叩1 because
[ι8]. Thus， se酷 onal
R eceived : D ecem ber 1 5， 2009
A ccep ted : M arch 22， 2010
P u b lish ed o n lin e in J・ STAGE: Aprill4， 2010
。
C orrespondence: Y F止凶作一冊a i l : fu k凶@ o b ih iro .ac.jp )
'2 0 1 0句 th eSoc間 企yfo r Reproduction and D evelopm ent
0- Reprod. Dev_ 56・ 39ι 399， 2010)
changes 00 in vitro em bryoni c devel opm ent of in vitro m atu田d
porcine oocytes follow ing ICSI throughout 由eyea仁
M . teri a I s and M et hods
The p 問 sent study was approved by th e Ani m al Experim ental
Com m i ttee of O bihiro U niversity of Agri cuI tu re and Veterinary
M edi ci ne in accord叩 ce w ith th e G ui di ng Prin ciples fo r th e Care
and Use ofResearch Anim als
Experim ental p erio d and tem peratu res in O bihiro
The present study was perform ed from Septem ber 2008 to
August 2009. The m onthl y tem perature data in O bihiro (4Z055' N，
143012'E)， H okkai do， Japan， we問 。btained 仕o mth e Japan M eteo-
ro lo gical Agency (h ttp ://八 w w w .jm a.go.jp/jm a ! indexe.h回 1 ).
O ocyte co lle ctio n
O varies w ere obtained from p 回 pubertal g ilts a t a Io cal slau gh ter-
house，
and transported to由elab o問to ry in a sterilized salin e (0. 9%
N aCI) so lu ti 叩 a t approxim ately 37 C w i 由in3 h and w ashed tw ice
in sterilized salin e so lu tio n . Cum ul us-oocyte com pl exes ( CO Cs)
w ere aspirated from 3 to 8 m m folIi cles using an 18-G needle
attached to a 5-m l disposable syrin ge. Subsequently，
w ashed th 悶 etim es in a H EPES-buffered Tyrode' s m edi um contain-
in g 0. 05% (w /v) polyvinyl alcohol ( PVA; Sigm a-Aldrich，
M O ， USA; TLH- PVA) . COCs w i 血tw oor 血ree layers of cum ul us
ce lls and unifonn cytoplasm w ere selected fo r I VM cu ltu re
th e COCs w ere
St . Louis，
In vitro m aluralion (IVM )
The m edi um fo r I VM w as M edi um 199 (w i 由 Earle's sa lts， L-
glutam ine， and 2， 200 m gl l sodi um bicarbonate; Si gm a) supple-
m ented w i 由 0. 05%PVA， 3.05 m M glucose ( W ako Pure Chem i cal
Indus岡 田 ， 口saka， Japan)， 0.91 m M N a-pyruvate (W ako) ， 100， uM

Page 3

SEASONAL CHANGES OF PORC llぜEI VP
397
cysteam i ne (Si gm a)， 10 ng/m l epidennaI grow th factor (Si gm a)，
叩 d75 mg ! 1 k叩 am yci n(Sigm a). Selected COCs w ere w ashed th ree
tim es in I VM m edi um ，
and由en 10- 15 COCs w ere cu ltu r吋 i na 100
μdropl et ofthe I VM m edi um covered w ith m ineral o iI (Si gm a) 白r
44 h a t 39 C in a hum idified a出l Osphe問 of 5 % C O2 in aIr. In th e
百 四t 22 h of culture， COCs w ere cultured w ith 10 IU /m l P MS G
( ASKA Phannaceutical ，
Tokyo，
Japan) and !O J U /m l hCG ( ASKA) ，
叩 din th e la st 22 h，
they we問 CllI tU田 dw ithout these honnones
In traCljlopla5m ic 5penn in je ctio n (IC5I)
After I VM cu ltu問 ， 出e oocytes we問 denuded 骨o mth eir c 九 l ffi U-
Iu s cells by gent J e pipetting in th e I VM m edi um . Denuded oocytes
、 ;verew ashed th ree tim 田 andkept in fresh I VM m edi um u n til ICSI
As in ourp 問vious studies [1-3]，
th e sam e batch ofpeI l eted frozen
sem en derived 世間1ejaculates of a Duroc boar was used through-
out th e study. The frozen sem en w田 thaw ed in p田wanned (39 c)
D ul becco' s phosphate buffered saline ( PBS; Gi bco- BRL， Grand
Island， N Y， USA) contai ni ng 0 .1 % P V A ( PBS- PV A) and was
w ashed by centrifugation a t 300 x g fo r 3 m i n in th e sam e m edi um
The procedure fo r ICSI was per品nned as previously reported
[13] w i th sligh t m odifications. Brie日 y， m ani pul ati on was con-
ducted w ith the aid of a pair of m i crom ani pul ato目 (Leitz， 九 lIe包la巳
Gennany) under an inverted m i croscope. Several drops of PBS-
PVA( 5μ1 each) containing spennatozoa 血 dTLH- PVA containing
denuded oocytes we回 placed on th e lid of a 50 x 9 mm p etri dish
(Falcon 1006， Franklin L古 田 ， NJ ， USA) and covered w ith m i nerョ l
o i l . A 1- μsupem atant portion of w ashed sem en w as added in to
PBS- PV A on a m ani pul ati on d ish . An injection pipette was p re-
pared by breaki ng the tip of a pulled capillary tube (G - 1 00;
N arishige，
Jap剖 ) w ith an inner diam eter 3- 4 J1 l1 l. From th e
edge of th e spenn-containing droplet， a m otile spennatozoon was
aspirated in to th e in jection pipette ta il-first w ithout an i m m obi l i z四
ing treatm ent， such as tail-scoringlcutting， and transferred to the
drop containing oocyt 田 Anooc戸e w 田 heldw ith its polar body a t
白 血e r th e 6 o r 12 o ' c 1ock position using由eholding p ip ette. A sper-
m atozoon was injected in to the oocyte cytopl asm and m i xed w ith
th e cytoplasm ic com ponents by gently sucking and bl ow i ng back a
sm all p art of cytopl asm using a m outh regulated open tube
Tokyo，
In vitro cu/tu re (IVCJ
After ICSI，
com pri sed of Porcine Zygote M edi um - 4 [14] suppl em ented w ith
2. 77 m M m yo-in ositoI ( Si gm a)， 0.34 m M tri-sod山 m citrate
( M erck， Darm stadt， Gennany) and 5 0 μ M s - m ercaptoethanol
(Sigm a). Im m edi atel y after w ashi ng，
cultured under 5 % C O2，
of l VC rnedi um cove陀 dwi 由m i neral oi l .
oocyt目 、.ve re w ashed three tim es in I VC m edi um
groups of l(ト1500cyt田 we問
5 % O2叩 d90% N2 a t 39 C in a 30 p l drop
O bservations o j m aturation，
b la5 to cy5 t5
In th is study， 叩 oocyte w ith an ex回 ded first polar body was
conside問 d 1 0 be m atu陀 A 宜er 12 h ofI CSI， som e oocytes we問
fixed in 25% (v/v) acetic acid ( W ako) in ethanol ( W ako) fo r 24 h
叩 dstained w i 出 1 %(w /v) orcein (Si gm a) in 45%a田 tic acid solu -
tio n fo r th e detennination of fertilizatio n rates. O ocytes 、 吋 出 m al e
and fem ale pronuc 1e i ， tw o polar bodies and a spenn ta il we問 con-
je rtiliza t 閉じ c Ieavageand
si dered to be norm al 1y ferti1 i zed. The rates of c 1eavage and
blastocyst fonnation w ere detennined a t 48 叩 d 144h仕' omthe sta rt
ofI V C，
respectively. T he num ber of ceI l s per blastocyst was deter-
m i ned by staining w ith 5 pgl m l H oechst 33342 (Si gm a) fo r 15 m i n
and using a fluorescence m i croscope
Experim ental d ， 国 松n
In th is study， when the m onthl y m ean tem peratures w ere less
than 0 C， it was considered to be w inter ( Decem ber， January， and
February)，
and th e rem ai ni ng seasons w ere c 1assified in to the fo l-
l ow i ng three rnonths in tervals: M arch to May (spring)， J une to
August (sum m er)，
and Septem berto Novem ber (autum n). The ra1es
of m aturation， 告 はilizatio n， c 1eavage and blastocyst fonnation w ere
calculated fo r each m onth of th e year and grouped in to品urseasons
to detennine th e effect of seasonal changes on in v itro devel opm en-
ta l com petence of porci ne oocytes. Thereafter， the correlation
betw een m onthl y m ean tem peratures and devel opm ental com pe-
tence of po四 in e oocytes was investigated
5 tat副 四1an alY5お
The experi m ent was repeated a t !east four tim es per m onth. Sta-
tistica l analyses w ere perfoロ nedusing SAS so仕 wa問 ( SASIn stitu te，
Cary， N C， USA) . The rates ofm aturati on， c 1eavage and blastocyst
fonnati 叩 w ere analyzed by lo gistic regressi叩 お 1 I0w i ng a bi nom i -
n a l. d istrib u tion using 由e fol l ow i m g m odel : In ( α / 1-<<)=s 十 四m
白 目 。r (seasons)， w hereα =the frequency of positive outcom e and
かthe intercept . The com pari son of blastocyst cel I num bers w田
conducted by one-w ay ANOVA. Post-hoc an alysis w田 perform ed
by Tukey- Kram er m ultiple com pari son te sts to detennine sign ifi-
cant differences. The correlation bet ween the atm ospheri c
tem perature and em bryoni c devel opm ent was analyzed by Pear
son's product-m om ent coefficient of co町芭latio n . D ifferences w ere
considered sign ifican t when出eP value was less 白血 0.05.
Re5ul ts
1 n 出155加dy， 5646 oocyt 回 、 問 問 used fo r I VM血 d 2752 oocyt田
we田 insem inatedby ICSI . As a w hol e，
throughou1 th e year. 百.e e偽 cts of seasonal changes on m eiotic 叩 d
developm ental com peten田 inporcine ooc，
and2，目sp ectively. The rates of m aturation 皿 d fe rtilizatio n we問 not
sign ifican tly d i fT erent am ong th e four seasons. The m ean c 1eavage
ra te in sum m er (75.7% ) w田 5 1伊 i自 C叩 t1y (P<0.05) higher than in th e
other seasons (60.7- 67.3% ; Table 2 )，
tence up to血e blastocyst stage w出 sign ific曲 目Y (P<0.05) high町 in
w i nter(31.1% ) 出回 inth e other seasons (19.7-23.5% ). Furtherm o同
th e m ean cel I num ber per blastocyst produced in autum n (43.8) w出
5ignificantly (P<0.05) higher th叩 those (25.6ー 27.9) in the other
seasons (Table 2 ). A significant (P く0.05) 叩 rrelation was 日 ound
betw een the m onthl y rnean blastocyst rates and the tem peratures
throughout 血e year (r=-O .5944，
not detected betw een the m ean tem peratures and cleavage rates
421 b lastocysts we陀 produced
戸e 5 are 曲 目 叩 inTables 1
and th e developm entaI com pe-
Fig. 1 ); how ever，
correlation was
Di scussi on
The in vitro devel opm ental com petence of porcine oocytes is

Page 4

! 3 0 0 2
; 川i
」 -jillii
SUZUKI et al
or the subsequent d.evelopm ent of bovi ne em bryos. The r 田 son fo r
these contradictory findings is not clear， but at least， the present
study has indicated th at the seasonal changes af : 晶c t porci ne I VP
perfoロ nance. Oo 血e other h阻 止 in the present study， 由e m atura-
tio o rates we問 oot significantly different am ang the four seasons，
but the b l 田 tocyst fonnati on w田 variable am ong seasons. It has
been reported血a t subjecting porcine ovari 田 to e!evated tem pera
tu悶Sof 41- 42 C does sot affect the m eiotic com pete 泊ceof oocytes
but decreases 血esubsequent devel opm ental com 戸tence [9 ]. Al so，
there are som e 田 poロ s th at heat stress i nduces detrim ental effects
on porci ne oocyte quality [7-9]. From these resu l t ! 写
that seasonal changes do sot directly affect nucl ear m aturati on
rates， w hereas the oocyte quality including cytopl asm i c m aturation
m ay be i m pai red for subsequent deve J opm ent . In other w ords，
sonal variations in the quality of oocytes affected the yields of
blastocysts，
c 1eavage rate in sum m er was significantly hi gher 由回 in the other
seasons. The reason fo r the high c 1eavage rate in sum m er is not
c 1ear; how ever，
the 目te of devel opm ent of blastocys15 to cleaved
em bryos was l ow est in sum m er com pa問 dw i th the other seasons in
th is study. Thi s resu lt m ay have been caused by sum m er heat stress
and suggests th at porcine em bryos produced duri ng sum m er m ay
， we concl ude
sea-
as reported in bovi ne l VF [21]. In the pres叩 tstudy，
the
気'-0
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398
stiII l ow er com pared w i th 由a t of in vivo produced em bryos [15，
16]，
although m any effo rts [17] have been m ade to i m prove porcine
I VP technologies. The present investigation of porci ne l VP ef:日 -
ci ency clearly dem onstrated that the in vitro devel opm ental
com petence of porcine oocytes up to th e blastocyst stage w田 d iι
ferent am ong seasons， w i th the highest efficiency occurri ng In
w inter. Addi ti onal l y， a negati ve correlation betw een the atm o-
spheric tem peratures and the blastocyst rates was observed in the
present study. These 阻 su l15 suggest th at one of the dom i nant fac-
岡 田 determ i ni ng 由e in vitro em bryoni c devel opm ent of porci ne
I VP is the m onthl y atm ospheri c tem perature.
In th is study，
the stock solution fo r sam pl i ng of ovaries was sta-
bi l i zed at approxi m atel y 37 C， and cul ture of oocytes was
per品 ロneda t the sam e tem pera加問 throughout the year. There品 目 ，
the m onthl y differences of in vitro devel opm ental com petence of
porcine oocytes throughout the year， 田 sho、 四 回 th is study，
m ost likely due to seasonal changes， including the atm ospheri c
tem perature. I t has been 悶 poロ ed th at surnrner heat stress reduces
th e devel opm ental com petence ofbovi ne oocytes [6 --8 ]. Addition-
ally，
heat stress was found to change th e num bers of fo llicles 血 d
th eir diam eters [6 ] and also to m odi かthe fatty acid [6 ] and protein
[18] com posi ti on ofthe porcine oocyte pl asm a m em brane. Further-
m ore，
heat st問 ss i nduces reactive oxygen species and results in
ap叩to sis of granulosa cells in m i ce and raω[ 19， 20]. As reported
in previous bovi ne studies [1ι8]，
th e present resu l15 c 1early show ed
a negative 問 lationship betw een devel opm ental com petence ofpor-
cine oocytes and atm ospheri c tem pera岡 田s
In contrast， Silva e t al . [21]
in vilro m atured bovi ne oocytes were signi 日cantly hi gher in
autum n than other seasons fo r a period of t wo years. Furtherm ore，
Ri vera e l a l. [22] indicated th at season did not affect cl eavage rates
we問
問 ported th at 出e blastocyst rates of
Tabl e t . E町田tof seasons on in vitro m aturation ra te in po悶 in e oocytes
主. ofo笠 笠
M atured ( 出 土 SEM )
Season (C )ホ
C ultured
The m onthly m ean b lasto cyst rate s
tem peratures from Septem ber 2008 to August 2009 in O bihiro，
H okkaido. Jap an . W hite b ars show m ean b lasto cyst ra te s in each
m onth. and black square and w hite trian gle I i n es in d icate th e
m onthl y m ean and m axi m um atm ospheri c tem peratures，
respectively. A sign i 日 cant (P<O .05) correlation w as found
betw een th e b l 田to cyst ra te s and t e m p e 同
and a伽lo sp h eric
国同s(~. 5 9 4 4 )
M O lltll
841 (73.3 土2. 1)
588 (70.3 土3.1 )
1479 (69.6土 1. 8 )
10% (71 .3 士2 .6 )
1147
837
2124
1538
Spring (6 .3 )
Sum m er (17.5)
Autum n (10.5 )
W inter(-3.8)
Fig. 1 .
' " Seasons w ere .cla ssifie d by th e m onthly m ean tem p eratu res. Spring is
M arch to M ay. sum m er is June to August ，
N ovem ber and w inter is Decem ber to Februa叩C : m ean tem pera仙re s of
th ree m onths each

autum n is Septe 四 b er to
副asto cysts/
c 1eaved em bryos:
出土SEM
34.9土4.8.
28.2:t 3.3 a
32.5 :t 3.6 a
47.9:t 3 .2 b
C ellslb l 田to cyst
m ean土SEM
Table 2 . Effect ofs田 sons on in vifro fe rtiliza tio n and em bryonic developm ent fol J ow i ng ICSI in p orcin e oocytes
Seぉon(C )・
B lasto cysts
{ 日 土SEM )
N o. of oocytes
C u ltu ば .e d Cleaved
( 出 土SEM )
N o. of oocytes
Exam i ned Fertilized
( 出 土SEM )
26.5 :t 1 . 8 •
27.9土2.6.
43.8土6 .2 b
25.6土 1. 2a
94 ( 23. H 2.8)'
7 1 (21 .3 土2 .6 ) •
117 (19.7土2 .4 ) •
139 (31.1土2.3 ) ，
269 (67.3004.2 )'
252 (75.7土2 .7 ) ，
360 (60.7土2 .6 ) b
290 ( 64. H 2 .8 ) . ，
0
3
3
7

0
3
9
4

4
3
5
4
125 (58. H 3. 7)
72 (52. 2土 5. 4)
170 (55.6 B. 5)
177 (55.0土4 .7 )
3
8
6
2

1
3
0
2

2
1
3
3
Spring (6 .3 )
Sum m er (17.5)
Autum n (10.5 )
W inter (-3.8)
日 Values w自 由 d ifferen t su p erscrip ts w ith in th e sam e col um n a re sign ifican tly d旧町.e n t (P<0.05)
tu re s (Fig. 1 ). Spring is M arch to M ay. sum m er is June to August ，
atu res ofthree m onths 田 ch
キSe田ons w ere c 1assified by th e m onthly m ean tem pera-
autum n is Septem ber to N ovem ber and w inter is D ecem berto Febru ary. C : m ean tem per-

Page 5

5EA50NAL CHANG E5 OF PORCI NE IVP
399
bem o 問 susceptible to fragm entation山田 those produced in other
5 e田 on5. Furthennore， W ol fenson e t a l. [23， 24] and Roth e t al . [25，
26] reported that foI l icular steroid producti on was reduced in
autum n by a caπ ")'-over effect of sum m er heat stress and陀 sultedin
a reduced dom i nance of th e selected fo lli c 1es and af晶cted bovi ne
oocyte quaI i ty . In白ct， 由e c J eavage 叩 dblastocyst rates in autum n
tended to be l ow er com pared w ith those ofthe other seasons in由e
p問sent study
百lere w田 a sign ifican t seasonal d i f:島 問n田 in th e ce ll num bers
per blastocyst produced in our stu d y. The m ean ce ll num bers per
blastocyst produced in autum n w ere higher 由an those in th e other
seasons. Al though we co .u ld oot c 1a rifシ 皿y 田氏ain reasons fo r 血e
distinction of the cell num bers per blastocyst exam i ned in the
present study， it is possible th at high-quality oocytes devel op to
blastocysts w ith a high ce ll num ber， w hereas low -quality oocytes
fa il to devel op to blastocysts under sum m er heat stress
百lepresent study was perfonned in H okkai do in northem Japan，
w hi ch has a com paratively colder clim ate 出an m ai nl and Japan. lt
has been 問 ported in pigs th at seasonal in fertility associated w ith
atm ospheric tem peratures is observed in B ritain，
15 田 cold 田 inHokkai do [2 7 ]. Fuはhe的 ore， an environm ental tem -
pe悶 tu re range of 18 to 20 C has been found to support op tirn al
reproductive perfonnance of grow ing-finishing pigs 加 d 白紙 signs
of heat s t問 ss occur a t above 22 C [2 8 ]. AdditionaI l y， th e m onthl y
m axi m um atm ospheri c tem peratures duri ng the sum m er season
、回目 over 30 C in O bihiro， H ol d 咽 id o， Japan (Fig. 1 ). How ever， not
only th e environrnentaI tem pe問 回 目: s but also th e hum idity， photo-
period and feeding system s are 回 sponsible fo r seasonal effects on
reproduction [2 9 ]. The問自ore， rno問 infonnation about seasonal 白c .
to rs influencing I VP efficiency is defin itely needed. The present
resu lts clearly dernonstrated白紙由eatm ospheric tem peraむ u re IS one
of th e dom i nant facto目 influencingon blastocyst production， w hi ch
provides valuable inform ation fo r 由e fu tu re study ofporcine IVP.
In conclusion， 由e p田'se n t 問 su lts show ed 出a t porcine em bryos
could be produced throughout th e year， but th at 出eI VP efficiency
was significantly affected by season， i.e . ， atm ospheri c tem pera
tu res. Furthennore， the resu lts show ed th at w inter is a favorable
season fo r blastocyst production in the region of O bi hi ro， Hok-
kaido，
Japan
whe回 出e clim ate
Acknowl edgm ent s
The autho 目 thank Dr K Ni wa， O kayarna U niversity， Japan， fo r
supplying froz叩 boar sem en. We also thank the Hokkai do M eat
Board in O bihiro fo r supplying po 四 in e ovaries. H W is supported
by J SPS Re5ea叩 h Fellow ships forYoung Scien tists
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創w
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